An ever-increasing number of human genetic diseases are attributed to expansion of trinucleotide repeats (TNRs). For example fragile X syndrome, the second leading cause of mental retardation, is due to expansion of a CGG tract and myotonic muscular dystrophy, is due to expansion of a CTG tract. Moreover, expansion of CGG tracts induce breakage at five known human chromosomal loci, some of which correlate with human disease. Chromosome breakage at fragile site is also implicated in the chromosomal rearrangements characteristic of human tumors. TNRs associated with human disease are rare. TNR and fragile site research would be greatly aided by genetic assays for tract expansion and/or chromosome fragility. The goal of this grant is to develop Saccharomyces cerevisiae as a model for TNR expansion and triplet-mediate chromosome fragility. The investigator has inserted 130 repeats of CTG onto a yeast chromosome. Large expansion of this tract were obtained, the first large expansion reported outside of humans. CTG tracts may also be length-dependent fragile sites in yeast. This observation provides the basis for a simple, genetic assay for tract expansion and chromosome fragility. The assays will be used to identify yeast and human genes whose mutation or over-expression increases CTG expansion and/or fragility. A premise of the proposed work is that most genes that affect TNR stability will encode proteins involved in replication, repair, or chromatin structure, many of which are conserved from yeast to humans. The behavior of CGG tracts will also be studied. Nucleases and DNA methylases will be used to analyze the chromatin structure of CTG and CGG tracts as both DNAs display unusual nucleosome forming properties in vitro. Finally, fragile sites are thought to be caused by late replication of TNR DNA. This model will be tested using density transfer and two-dimensional gel electrophoresis to determine if replication timing or replication fork progression is affected by the presence of TNRs.